Revealing Brownish Mycena Diversity in China: New Discoveries and Taxonomic Insights

Within the genus Mycena, species exhibiting brownish basidiomata present considerable challenges in identification due to similar coloration. This study underscores the significance of pileipellis types and cheilocystidia characteristics as critical in delimiting brownish Mycena species. To clarify the principal taxonomic characters and their utility in distinguishing between brownish Mycena species, a morphological taxonomy and phylogenetic analysis were performed. Five new species from China were introduced and characterized through a comprehensive morphological anatomy and phylogenetic substantiation: M. campanulatihemisphaerica sp. nov., M. digitifurcata sp. nov., M. kunyuensis sp. nov., M. limitis sp. nov., and M. oryzifluens sp. nov. Discussions of these taxa are supplemented with morphological illustrations. The phylogenetic relationships were inferred using Bayesian Inference and Maximum Likelihood methods based on sequences from the internal transcribed spacer and the large subunit regions of nuclear ribosomal RNA. With the addition of these five new species, the worldwide count of brownish Mycena increases to 94, and a key to the 29 known species of brownish Mycena from China is presented.


Introduction
Mycena (Pers.)Roussel was established in the early 18th century, standing as one of the earliest genera among fungi [1].Currently, there are over 500 known species, with 2418 listed in the Index Fungorum (https://www.indexfungorum.org,accessed on 27 February 2024), highlighting a remarkable species diversity [2].Mycena species are widely distributed all over the world, and play a crucial role in the decomposition of dead branches, fallen leaves, and rotting wood, which facilitates the material circulation of forest ecosystems [3][4][5][6][7].Moreover, some species have demonstrated a propensity for enhancing Orchidaceae seed germination, which contributes to the resilience and diversity of ecosystems [8][9][10].The diverse morphology and color of Mycena contribute a high species diversity, while they also present significant taxonomic challenges due to subtle morphological variations and complex microstructures among species [11][12][13][14][15][16].Taxonomists and surface characteristics were detailed in records based on photographs and field notes.The nomenclature of color descriptions refers to Ridgway (1912) [33].The extent of pileus marginal striations or sulcations was quantitatively assessed by the ratio of the striation's or sulcation's length to the pileus's radius (n R) [34].The specimens were dried in an electric oven (Stöckli, A. & J. Stöckli AG, Netstal, Switzerland) at 40 • C and were then placed in a ziplock bag with allochroic silica gel for preservation.All specimens are now preserved in the Fungarium of the Fujian Academy of Agricultural Sciences (FFAAS), China.
Microscopical features were examined with a Lab A1 microscope (Carl Zeiss AG, Jena, Germany) using dried specimens rehydrated in a 5% KOH aqueous solution and stained with a 1% Congo red aqueous solution when necessary.Melzer's reagent was used to test the amyloidity of basidiospores and lamellar trama [35].Basidiospores were photographed using ZEN 2.3 (Blue Edition) software (Carl Zeiss Microscopy GmbH, Jena, Germany), with at least 20 basidiospores measured per specimen.For the holotype, a more extensive examination entailed measuring 40 or more basidiospores from each basidiomata.The measured results were presented in the form [a/b/c] (d) e-f -g (h) The abbreviation [a/b/c] represents a number of basidiospores measured from b number of basidiomata of c number of collections; d and h represent the minimum and maximum lengths (5% extremum), while the range e-g encompasses the remaining 90% of the measured values, with f as the mean length.Width (i-m) and Q values (n-q) are analogous representations.If the difference between d and e, g and h, i and j, or l and m is less than 0.2 µm, and the difference between n and o or p and q is less than 0.01 µm, d, h, i, m, n, or q were omitted accordingly.Q represents the "length/width" ratio of a basidiospore.Q m presents the average Q of all basidiospores ± the sample standard deviation [36].Other microstructures examined include basidia, cheilocystidia, pleurocystidia, pileocystidia, caulocystidia, and the respective compositions of the pileipellis and stipipellis, along with lamellar trama.For each specimen, 20 of these structures were recorded, detailing their sizes, shapes, contents, and other features.The pileipellis samples were specifically cut from the mid-zone between the center and the margin of the pileus, while stipitipellis samples were procured from the middle of the stipe.
The delineation of macro-and microstructures commenced with manual sketches on A4 paper using a pencil and subsequently transposed to tracing paper using a Sakura needle pen (Sakura Color Products Corporation, Osaka, Japan).The sketches were converted to digitalization in TIF format using a Canon LiDE120 scanner (Canon, Tokyo, Japan).Photoshop was then utilized for further refinement and typesetting.

Molecular Phylogeny
The newly obtained sequences were subject to BLAST searches within the NCBI GenBank database (https://www.ncbi.nlm.nih.gov/,accessed on 27 March 2024) to retrieve and download homologous sequences (with nucleotide identities >90%).Furthermore, sequences of species bearing a morphological resemblance to the species in this study were also sourced from GenBank (https://www.ncbi.nlm.nih.gov/genbank/,accessed on 27 March 2024), selecting one or more representatives per species that share a morphological or phylogenetic affinity with the newly described species for preliminary phylogenetic analysis.M. tenuispinosa J. Favre and M. mucoroides Aronsen were used as outgroup taxa to the root tree.Gaps were treated as missing data after aligning the ITS and LSU datasets independently using the auto strategy (FFT-NS-1, FFT-NS-2, FFT-NS-i or L-INS-i) by MAFFT v.7.110, with gene fragments manually refined and amalgamated in MEGA 5 [44][45][46].Both Bayesian Inference (BI) and Maximum Likelihood (ML) were utilized for phylogenetic inference.For BI analysis, the optimal nucleotide evolution was estimated using Modeltest 2.3 with the BIC criterion [47].MrBayes 3.2.6 executed the analysis over 5,000,000 MCMC generations; four chains were conducted with the sampling every 500th generation.The first 25% of trees were discarded as burn-in after the average standard deviation of split frequencies under 0.01, with results aggregated through the "sump" and "sumt" commands [48].Tracer v.1.7.2 served in the visualization and examination of MCMC trace files from the Bayesian phylogenetic inference [49].Meanwhile, ML analysis was conducted by RAxMLGUI 2.0 with the GTRGAMMA model and 1000 bootstrap (BS) replicates using default parameters [50].Finally, the phylogenetic tree was visualized in FigTree v.1.4.3 and refined in Photoshop CS4.

Phylogenetic Analysis
The molecular analyses dataset consisted of 152 sequences in total, including 28 newly obtained sequences (14 spanning ITS and LSU) and 124 sequences downloaded from GenBank (98 ITS and 26 LSU).Detailed information is provided in Table 1.The aligned dataset culminated in a total of 1743 aligned sites, including gaps (824 ITS, 919 LSU).The average deviation of split frequencies was 0.006, the ESS (effective sample size) was 1908.3, and the average Potential Scale Reduction Factor (PSRF) parameter values = 1.000 after 5,000,000 MCMC generations.The ML phylogenetic analysis yielded a final loglikelihood value of −13,761.724187.The average bootstrap value was 78.5%, indicating a generally high confidence in the tree's branches.The lowest bootstrap value was 50.0%, suggesting some branches may require further verification, while the highest value was 99.0%, reflecting very high confidence in certain branches.The tree topology effectively depicted the evolutionary relationships among the major taxa, with samples from each novel species clustering together as expected.Remarks: New generated sequences are emphasized in bold; "-" show missing sequence.
The phylogenetic evaluations using ML and BI indicated generally similar topologies for the five newly identified species, although some discrepancies between the methods were observed.Consequently, the BI tree was selected to be shown in Figure 1, incorporating only those nodes supported by bootstrap values of at least 75% and posterior probabilities exceeding 0.95.The phylogenetic structure has resolved into 13 distinct clades, each delineated by shared morphological characteristics of pileipellis hyphae or cheilocystidia among the species within them.While some clades received lower support rates, the topology of the phylogenetic tree still effectively reflects the shared characteristics of groups within these clades, such as smooth or ornamented characteristics.
The five newly identified species demonstrated high support within the phylogenetic tree, with each being robustly placed within clades 1, 2, 9, and 10.These results reinforce the taxonomic standing of these clades and confirm the systematic placement of these new species.Clade 1 emerges as a monophyletic clade, incorporating M. oryzifluens sp.nov.supported by three specimens (FFAAS1051, FFAAS1052, and FFAAS1053).In the BI phyloge-netic reconstruction, M. oryzifluens sp.nov.clusters closely with other members of Clade 1, indicating a distinct genetic lineage.Although in the ML analysis, M. oryzifluens sp.nov.and M. amicta (Fr.)Quél.span a broad clade from Clade 2 to Clade 13, their monophyletic status is firmly supported (BS/BPP = 100/1.00).Additionally, species in Clade 1 share the characteristic of non-smooth pileipellis hyphae.Clade 2 incorporates the new species M. digitifurcata sp.nov.(FFAAS1054, FFAAS1055), all members sharing the feature of non-smooth pileipellis hyphae.In the BI tree, M. digitifurcata sp.nov. is closely related to M. cristinae, although this relationship is not as strong in the ML analysis.Despite variations between the BI and ML trees, M. digitifurcata sp.nov. is consistently recognized as a monophyletic entity with high statistical support (BS/BPP = 99/1.00).Species of Clade 3 exhibit smooth pileipellis and cheilocystidia, receiving unanimous support (100%) and demonstrating high phylogenetic consistency.Clade 4, although supported at a lower rate (BS/BPP = 81/1.00),presents species with cylindrical excrescences on the cheilocystidia, providing key taxonomic insights.Clades 5 and 6 both exhibit very high support rates (BS/BPP = 100/1.00),where Clade 5 species feature smooth cheilocystidia, while Clade 6 species display excrescences on the cheilocystidia.Clades 7 and 8 also showcase specific morphological characteristics, with Clade 7 having non-smooth pileipellis and Clade 8 featuring non-smooth pileipellis, along with some species having gelatinous layers.Clade 9 includes M. limitis sp.nov.(FFAAS1056, FFAAS1057, and FFAAS1058), sharing a close phylogenetic relationship with M. niveipes (Murrill) Murrill, along with other species such as M. seynii Quél., M. bulliformis B.A. Perry & Desjardin, and M. fulgoris Cortés-Pérez & Desjardin.Although Clades 9 and 12 form independent groups in BI analysis, Clade 12 nests within Clade 9 in ML analysis.Nonetheless, the position of M. limitis sp.nov.remains well-supported (BS/BPP = 100/1.00).Clade 10 comprises the novel species M. campanulatihemisphaerica sp.nov.(FFAAS1047, FFAAS1048, and FFAAS1049) and M. kunyuensis sp.nov.(FFAAS1045, FFAAS1046), sharing characteristics of smooth cheilocystidia with other species.Clade 11, though not highly supported, includes species that exhibit smooth cheilocystidia.Clade 12 is fully supported (100%), with species displaying smooth cheilocystidia and non-smooth pileipellis, showing clear phylogenetic consistency.Clade 13, similarly supported fully (100%) in the BI tree, shares the characteristic of non-smooth pileipellis among its species.
MycoBank no: 853739 Etymology: The epithet campanulatihemisphaerica combines the Latin feminine adjectives campanulata (bell-shaped) and hemisphaerica (half-spherical).This name accurately describes the shape of the pileus, which transitions from bell-shaped to nearly hemispherical.
Habit and habitat: Fascicled or scattered on rotten branches in mixed broadleaf-conifer forests, mainly under trees of Picea, Pinus, and Quercus.
ranges from black to dark brown, and its gelatinized pileipellis [23,28].Comparatively, M. kunyuensis and M. campanulatihemisphaerica exhibit similarities in some macroscopic and microscopic characters.However, M. campanulatihemisphaerica can be distinguished by its shorter fibrils-covered stipe; fuscous brown, markedly sulcate pileus, with its margin being slightly serrulate; larger basidiospores; and cheilocystidia that fork at the apex (Figure 8).MycoBank no: 853783 Etymology: The epithet oryzifluens is derived from the Latin word oryzi, meaning rice, and fluens, meaning flowing.This name reflects the rice-like appearance of the white, pruinose, and pubescent stipe and connects to the folklore of the type locality, where a legend speaks of a flowing rice cave, symbolizing abundance and continuity.MycoBank no: 853784 Etymology: The epithet digitifurcata derives from the Latin words 'digitus', meaning 'finger', and 'furcatus', meaning 'forked'.This name is chosen to describe the distinctive finger-like and forked projections at the apex of the cheilocystidia.
Diagnosis: Pileus deep gray when young, light drab to drab with age, basidiospores ellipsoid to narrowly ellipsoid, cheilocystidia with finger-like branches at apex.Differs from M. cristinae by pruinose pileus, obviously decurrent lamellae, and being weakly intervenose.
Habit and habitat: Scattered on rotten branches in mixed broadleaf-conifer forests, mainly under trees of Liriodendron, Pseudolarix, and Pinus.
Notes: M. cristinae closely resembles M. digitifurcata in pileus and stipe color, but it is distinguished by its smooth pileus, adnate lamellae, and markedly intervenose lamellae [26].M. pasvikensis Aronsen, sharing a similar pileus color with M. digitifurcata, is differentiated by a densely fibril-covered stipe base, serrulate lamellae, and gelatinized pileipellis and stipitipellis [28].Similar cheilocystidia shapes are observed in M. pseudopicta (J.E.Lange) Kühner and M. cinerella (P.Karst.)P. Karst., but M. pseudopicta is differentiated from M. digitifurcata by its gelatinized pileipellis and stipitipellis, and by possessing larger basidiospores [23,28]; M. cinerella is notable for its gelatinized pileipellis and the lighter color of the pileus [28,29].Microscopic observation reveals a variation in the branch lengths of the cheilocystidia.In specimen FFAAS1054, most cheilocystidia display short (0.9-1.9 µm) branched excrescences at the apex, whereas in FFAAS1055, cheilocystidia with significantly longer (7.5-22.2µm) branched excrescences at the apex are typically observed, indicating the various forms of cheilocystidia.MycoBank no: 853786 Etymology: The epithet limitis is derived from the Latin word limes, which emphasizes the morphological similarities with related species and highlights the limited distinguishing features that set this species apart from its close relatives.Additionally, this name pays tribute to the companionship and support provided by a forest ranger, affectionately called brother on the border. of species diversity [11][12][13][14]21,23,28]. Specifically, M. campanulatihemisphaerica, M. limitis, and M. kunyuensis demonstrate close affiliations with M. abramsii, M. algeriensis Maire, M. galericulata, and M. maculate P. Karst., all of which are endemic to China [21].Notably, M. campanulatihemisphaerica, M. kunyuensis, and M. abramsii exhibit a similar basidiomata coloration, with M. kunyuensis frequently misidentified as M. abramsii due to the analogous coloration of the pileus before morphological anatomy [21,23,28].Similarly, M. limitis is indistinguishable from M. algeriensis, M. galericulata, and M. maculata when identification is based solely on the color of the basidiomata [21,28].Moreover, species within the complex exhibit a comparable pileus coloration and are subject to variations due to changes in growth period and environment conditions, further complicating the accurate identification of these brownish Mycena species [11][12][13][14][69][70][71][72][73][74][75][76][77].For example, the M. filopes complex, as delineated by Arnolds (2015) and Aronsen (2016), is a case in point.[28,78].A parallel example is found in the M. pura complex.While basidiomata coloration has historically served as a criterion for subsection classification within the M. pura complex, recent insights by Liu (2023) suggest that the characteristics of the cheilocystidia and pleurocystidia, as well as the presence or absence of pleurocystidia, are critical for species differentiation [11,12,14,79].Consequently, morphological anatomy is an effective method for the identification of species within the brownish Mycena group.
Pileipellis types and cheilocystidia characteristics are integral to the delimitation of brownish Mycena species.In the taxonomic framework proposed by Smith (1947) and Maas Geesteranus (1992a, 1992b), pileipellis types and cheilocystidia characteristics are pivotal for sectional categorization [11,12,20].These characteristics are also crucial criteria for distinguishing various species of brownish Mycena, a delineation corroborated by molecular systematics.The phylogenetic tree constructed in this study is divided into 13 clades, and while some clades have lower support rates, the conclusions drawn from the phylogenetic tree remain consistent with those from the morphological anatomy analyses.Notably, M. campanulatihemisphaerica, M. limitis, and M. kunyuensis exhibit smooth cheilocystidia, and cluster phylogenetically into a clade and align closely with the species of sect.Fragilipedes, which also exhibit smooth cheilocystidia [17,28].Moreover, phylogenetic analyses reveal a distinct clade comprising M. digitifurcata, which aligns closely with the species of sect.Rubromarginatae [26,57].Sect.Rubromarginatae is characterized by marginta lamellae, ranging from deep yellow to dark greenish [11,12].If the sectional division relies solely on this feature, M. digitifurcata would ostensibly not qualify for inclusion within the sect.Rubromarginatae due to its lamellae not being marginated [11,12].However, its pileipellis types and cheilocystidia characteristics are congruent with those of the sect.Rubromarginatae, suggesting the need for a broader consideration of morphological characters.Accordingly, M. digitifurcata is classified within the sect.Rubromarginatae.This classification, proposed by Jadson (2021), identifies M. cristinae as a non-marginate lamellae member of the sect.Rubromarginatae [26].Our research aligns with Maas Geesteranus' (1992a, 1992b) interpretation of the sect.Rubromarginatae, which is further supported by the perspectives advanced by Na (2019) [11,12,21].Specifically, it emphasizes that the delimitation of sections exhibiting colored lamellae edges should predominantly consider variations in pileipellis types and cheilocystidia characteristics.Furthermore, pileipellis and cheilocystidia are present in almost all brownish Mycena, reinforcing their utility as key diagnostic features [23,28].For example, M. galericulata can be distinguished from its morphologic relative, M. algeriensis, by its tuberculated cheilocystidia, and M. leptocephala is distinguished from its close relative M. polygramma (Bull.)Gray by smooth pileipellis [11,12,21,23,28].Consequently, we reaffirm the role of pileipellis types and cheilocystidia characteristics as primary taxonomic criteria within the brownish Mycena group, corroborating the classifications of Smith (1947) and Maas Geesteranus (1992a, 1992b) and substantiated by the molecular systematics of Na (2019) [11,12,20,21].
Additionally, this study suggests that M. oryzifluens may represent a prospective novel section.Phylogenetically, M. oryzifluens is closely related to three established sections, namely: sect.Exornatae, sect.Cyanocephalae, and sect.Amictae.Despite these affiliations, M. oryzifluens exhibits distinct morphological characteristics that preclude its classification within these existing sections.These distinguishing features include a dry pileus, the absence of a blue disc at the stipe base, branched cheilocystidia apex, and non-gelatinized pileipellis [11,12,21,28,57].Consequently, M. oryzifluens does not align with any current sectional definitions and suggests the potential for defining a new section.However, due to the lack of enough specimen samples at present, it is prudent not to propose it as a new section within this publication.Future research efforts will collect and examine additional specimens to substantiate the distinctiveness of this taxonomic group.
At present, there are 24 brownish Mycena species in China.This study introduces 5 new species, increasing the total to 29.Our analysis of morphological characteristics has identified the types of pileipellis and cheilocystidia as critical distinguishing features for brownish Mycena.To facilitate future research and better species distinction, we provide an identification key for the brownish Mycena species in China.

Figure 1 .
Figure 1.Bayesian Inference tree based on concatenated ITS + LSU dataset.Only branch nodes with both Maximum Likelihood bootstrap support values above 75% and Bayesian posterior probabilities exceeding 0.95 are indicated.Red dots and text represent new taxa, black dots indicate the presence of both ITS and LSU sequences, and white dots signify the presence of only LSU sequences.

Figure 1 .
Figure 1.Bayesian Inference tree based on concatenated ITS + LSU dataset.Only branch nodes with both Maximum Likelihood bootstrap support values above 75% and Bayesian posterior probabilities exceeding 0.95 are indicated.Red dots and text represent new taxa, black dots indicate the presence of both ITS and LSU sequences, and white dots signify the presence of only LSU sequences.

Table 1 .
DNA sequences of Mycena used in the phylogenetic analysis in this study.